Diamine Beta2 Adrenergic Receptor Agonists

ABSTRACT

The invention provides novel β 2  adrenergic receptor agonist compounds. The invention also provides pharmaceutical compositions comprising such compounds, methods of using such compounds to treat diseases associated with β 2  adrenergic receptor activity, and processes and intermediates useful for preparing such compounds.

FIELD OF THE INVENTION

The invention is directed to novel β₂ adrenergic receptor agonists. Theinvention is also directed to pharmaceutical compositions comprisingsuch compounds, methods of using such compounds to treat diseasesassociated with β₂ adrenergic receptor activity, and processes andintermediates useful for preparing such compounds.

BACKGROUND OF THE INVENTION

β₂ adrenergic receptor agonists are recognized as effective drugs forthe treatment of pulmonary diseases such as asthma and chronicobstructive pulmonary disease (including chronic bronchitis andemphysema). β₂ adrenergic receptor agonists are also useful for treatingpre-term labor, and are potentially useful for treating neurologicaldisorders and cardiac disorders. In spite of the success that has beenachieved with certain β₂ adrenergic receptor agonists, current agentspossess less than desirable duration of action, potency, selectivity,and/or onset. Thus, there is a need for additional β2 adrenergicreceptor agonists having improved properties, such as improved durationof action, potency, selectivity, and/or onset.

SUMMARY OF THE INVENTION

The invention provides novel compounds that possess β₂ adrenergicreceptor agonist activity. Among other properties, compounds of theinvention are potent and selective β₂ adrenergic receptor agonists. Inaddition, an exemplary compound of the invention has been demonstratedto possess a surprising and unexpectedly long duration of action, whichallows for once-daily, or even less frequent, dosing.

Accordingly, this invention provides a compound of formula (I):

wherein:

each of R¹, R², R³, and R⁴ is independently selected from hydrogen,hydroxy, amino, halo, —CH₂OH and —NHCHO, or R¹ and R² taken together areselected from —NHC(═O)CH═CH—, —CH═CHC(═O)NH—, —NHC(═O)S—; and—SC(═O)NH—;

R⁵ is selected from hydrogen, —OR^(a), and —NR^(a)R^(b), wherein R^(a)and R^(b) are each independently hydrogen or C₁₋₃alkyl;

n is an integer of from 0 to 7;

m is an integer of from 0 to 5; and

each of R⁶, R⁷, R⁸, R⁹, and R¹⁰ is independently hydrogen or C₁₋₆alkyl;

provided that when m is 0, R⁵ is hydrogen;

or a pharmaceutically-acceptable salt or solvate or stereoisomerthereof.

The invention also provides a pharmaceutical composition comprising acompound of the invention and a pharmaceutically-acceptable carrier. Theinvention further provides combinations comprising a compound of theinvention and one or more other therapeutic agents and pharmaceuticalcompositions comprising a compound of the invention, one or more othertherapeutic agents, and a pharmaceutically-acceptable carrier.

The invention also provides a method of treating a mammal having adisease or condition associated with β₂ adrenergic receptor activity(e.g. a pulmonary disease, such as asthma or chronic obstructivepulmonary disease, pre-term labor, a neurological disorder, a cardiacdisorder, or inflammation), the method comprising administering to themammal, a therapeutically effective amount of a compound of theinvention. In particular, the invention provides a method of treatingasthma or chronic obstructive pulmonary disease in a mammal, the methodcomprising administering to the mammal, a therapeutically effectiveamount of a compound of the invention.

The invention further provides a method of treatment comprisingadministering a therapeutically effective amount of a combination of acompound of the invention and one or more other therapeutic agents.

The invention also provides a method of treating a mammal having adisease or condition associated with β₂ adrenergic receptor activity,the method comprising administering to the mammal, a therapeuticallyeffective amount of a pharmaceutical composition of the invention.

The compounds of the invention can also be used as research tools, i.e.to study biological systems or samples, or for studying the activity ofother chemical compounds. Accordingly, in another of its method aspects,the invention provides a method of using a compound of formula (I), or apharmaceutically acceptable salt or solvate or stereoisomer thereof, asa research tool for studying a biological system or sample or fordiscovering new β₂ adrenergic receptor agonists.

In separate and distinct aspects, the invention also provides syntheticprocesses and intermediates described herein, which are useful forpreparing compounds of the invention.

The invention also provides a compound of the invention as describedherein for use in medical therapy, as well as the use of a compound ofthe invention in the manufacture of a formulation or medicament fortreating a mammal having a disease or condition associated with β₂adrenergic receptor activity (e.g. a pulmonary disease, such as asthmaor chronic obstructive pulmonary disease, pre-term labor, a neurologicaldisorder, a cardiac disorder, or inflammation).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides novel diamine β₂ adrenergic receptor agonists offormula (I), or pharmaceutically-acceptable salts or solvates orstereoisomers thereof. The following substituents and values areintended to provide representative examples of various aspects of theinvention. These representative values are intended to further definesuch aspects and are not intended to exclude other values or limit thescope of the invention.

In a specific aspect of the invention, R¹ is halo, —CH₂OH, or —NHCHO.

In other specific aspects, R¹ is chloro, —CH₂OH, or —NHCHO; or R¹ is—CH₂OH or —NHCHO.

In a specific aspect, R² is hydrogen.

In a specific aspect, R³ is hydroxy or amino; in another specificaspect, R³ is hydroxy.

In specific aspects, R⁴ is hydrogen or halo; or R⁴ is hydrogen orchloro.

In a specific aspect, R¹ is —NHCHO, R³ is hydroxy, and R² and R⁴ areeach hydrogen.

In another specific aspect, R¹ and R² taken together are —NHC(═O)CH═CH—or —CH═CHC(═O)NH—, R³ is hydroxy, and R⁴ is hydrogen.

In another specific aspect, R¹ is —CH₂OH, R³ is hydroxy, and R² and R⁴are each hydrogen.

In yet another specific aspect, R¹ and R⁴ are chloro, R³ is amino, andR² is hydrogen.

In still another specific aspect, R¹ and R² taken together are—NHC(═O)S— or —SC(═O)NH—, R³ is hydroxy, and R⁴ is hydrogen.

In a specific aspect, R⁵ is hydrogen or —OR^(a). Representative R⁵values within this aspect include, but are not limited to, hydrogen,hydroxy, methoxy, and ethoxy.

In another specific aspect, R⁵ is hydrogen or —NR^(a)R^(b).Representative R⁵ values within this aspect include, but are not limitedto, hydrogen, amino, methylamino, dimethylamino, ethylamino, anddiethylamino.

In another specific aspect, R⁵ is hydrogen.

In a specific aspect, R⁷ is hydrogen.

In another specific aspect, R⁷ is methyl.

In a specific aspect, R⁵ is hydrogen

In another specific aspect, R⁵ is methyl.

In a specific aspect, R⁹ is hydrogen.

In another specific aspect, R⁹ is methyl.

In a specific aspect, R¹⁰ is hydrogen.

In another specific aspect, R¹⁰ is hydrogen.

In a specific aspect, n is 2, 3, 4, 5, or 6.

In another specific aspect, n is 4.

In a specific aspect, m is 0, 1, 2, or 3.

In another specific aspect, m is 1.

In one aspect, the invention provides a compound of formula (II):

wherein:

R¹ is —CH₂OH or —NHCHO, and R² is hydrogen; or R¹ and R² taken togetherare —NHC(═O)CH═CH— or —CH═CHC(═O)NH—;

n is an integer of from 2 to 6;

m is an integer of from 0 to 3;

or a pharmaceutically-acceptable salt or solvate or stereoisomerthereof.

Particular mention may be made of the following compound:

-   8-hydroxy-5-[(R)-1-hydroxy-2-(6-phenethylamino-hexylamino)ethyl]-1H-quinolin-2-one    where the chemical nomenclature conforms to that of the automatic    naming program AutoNom, as provided by MDL Information Systems, GmbH    (Frankfurt, Germany).

As illustrated above, the compounds of the invention contain one or morechiral centers. Accordingly, the invention includes racemic mixtures,pure stereoisomers (i.e. individual enantiomers or diastereomers), andstereoisomer-enriched mixtures of such isomers, unless otherwiseindicated. When a particular stereoisomer is shown, it will beunderstood by those skilled in the art, that minor amounts of otherstereoisomers may be present in the compositions of this inventionunless otherwise indicated, provided that the utility of the compositionas a whole is not eliminated by the presence of such other isomers.

In particular, compounds of the invention contain a chiral center at thealkylene carbon in formulas (I) and (II) to which the hydroxy group isattached. When a mixture of stereoisomers is employed, it isadvantageous for the amount of the stereoisomer with the (R) orientationat the chiral center bearing the hydroxy group to be greater than theamount of the corresponding (S) stereoisomer. When comparingstereoisomers of the same compound, the (R) stereoisomer is preferredover the (S) stereoisomer.

DEFINITIONS

When describing the compounds, compositions and methods of theinvention, the following terms have the following meanings, unlessotherwise indicated.

The term “alkyl” means a monovalent saturated hydrocarbon group whichmay be linear or branched or combinations thereof. Unless otherwisedefined, such alkyl groups typically contain from 1 to 10 carbon atoms.Representative alkyl groups include, by way of example, methyl, ethyl,n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl,n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl and the like.

The term “halo” means a fluoro, chloro, bromo or iodo.

The term “treatment” as used herein means the treatment of a disease ormedical condition in a patient, such as a mammal (particularly a human)which includes:

-   -   (a) preventing the disease or medical condition from occurring,        i.e., prophylactic treatment of a patient;    -   (b) ameliorating the disease or medical condition, i.e.,        eliminating or causing regression of the disease or medical        condition in a patient;    -   (c) suppressing the disease or medical condition, i.e., slowing        or arresting the development of the disease or medical condition        in a patient; or    -   (d) alleviating the symptoms of the disease or medical condition        in a patient.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need of treatment.

The phrase “disease or condition associated with β₂ adrenergic receptoractivity” includes all disease states and/or conditions that areacknowledged now, or that are found in the future, to be associated withβ₂ adrenergic receptor activity. Such disease states include, but arenot limited to, pulmonary diseases, such as asthma and chronicobstructive pulmonary disease (including chronic bronchitis andemphysema), as well as neurological disorders and cardiac disorders. β₂adrenergic receptor activity is also known to be associated withpre-term labor (see U.S. Pat. No. 5,872,126) and some types ofinflammation (see International Patent Application Publication Number WO99/30703 and U.S. Pat. No. 5,290,815).

The term “pharmaceutically-acceptable salt” means a salt prepared from abase or acid which is acceptable for administration to a patient, suchas a mammal. Such salts can be derived from pharmaceutically-acceptableinorganic or organic bases and from pharmaceutically-acceptableinorganic or organic acids.

Salts derived from pharmaceutically-acceptable acids include, but arenot limited to, acetic, benzenesulfonic, benzoic, camphosulfonic,citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic,hydrochloric, lactic, maleic, malic, mandelic, methanesulfonic, mucic,nitric, pantothenic, phosphoric, succinic, sulfuric, tartaric,p-toluenesulfonic, xinafoic (1-hydroxy-2-naphthoic acid) and the like.Salts derived from fumaric, hydrobromic, hydrochloric, acetic, sulfuric,methanesulfonic, xinafoic, and tartaric acids are of particularinterest.

Salts derived from pharmaceutically-acceptable inorganic bases includealuminum, ammonium, calcium, copper, ferric, ferrous, lithium,magnesium, manganic, manganous, potassium, sodium, zinc and the like.Particularly preferred are ammonium, calcium, magnesium, potassium andsodium salts. Salts derived from pharmaceutically-acceptable organicbases include salts of primary, secondary and tertiary amines, includingsubstituted amines, cyclic amines, naturally-occurring amines and thelike, such as arginine, betaine, caffeine, choline,N,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol,2-dimethylaminoethanol, ethanolamine, ethylenediamine,N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine,hydrabamine, isopropylamine, lysine, methylglucamine, morpholine,piperazine, piperadine, polyamine resins, procaine, purines,theobromine, triethylamine, trimethylamine, tripropylamine, tromethamineand the like.

The term “solvate” means a complex or aggregate formed by one or moremolecules of a solute, i.e. a compound of the invention or apharmaceutically-acceptable salt thereof, and one or more molecules of asolvent. Such solvates are typically crystalline solids having asubstantially fixed molar ratio of solute and solvent. Representativesolvents include by way of example, water, methanol, ethanol,isopropanol, acetic acid, and the like. When the solvent is water, thesolvate formed is a hydrate.

It will be appreciated that the term “or a pharmaceutically-acceptablesalt or solvate of stereoisomer thereof” is intended to include allpermutations of salts, solvates and stereoisomers, such as a solvate ofa pharmaceutically-acceptable salt of a stereoisomer of a compound offormula (I).

The term “leaving group” means a functional group or atom which can bedisplaced by another functional group or atom in a substitutionreaction, such as a nucleophilic substitution reaction. By way ofexample, representative leaving groups include chloro, bromo and iodogroups; sulfonic ester groups, such as mesylate, tosylate, brosylate,nosylate and the like; and acyloxy groups, such as acetoxy,trifluoroacetoxy and the like.

The term “amino-protecting group” means a protecting group suitable forpreventing undesired reactions at an amino nitrogen. Representativeamino-protecting groups include, but are not limited to, formyl; acylgroups, for example alkanoyl groups, such as acetyl; alkoxycarbonylgroups, such as tert-butoxycarbonyl (Boc); arylmethoxycarbonyl groups,such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc);arylmethyl groups, such as benzyl (Bn), trityl (Tr), and1,1-di-(4′-methoxyphenyl)methyl; silyl groups, such as trimethylsilyl(TMS) and tert-butyldimethylsilyl (TBS); and the like.

The term “hydroxy-protecting group” means a protecting group suitablefor preventing undesired reactions at a hydroxy group. Representativehydroxy-protecting groups include, but are not limited to, alkyl groups,such as methyl, ethyl, and tert-butyl; acyl groups, for example alkanoylgroups, such as acetyl; arylmethyl groups, such as benzyl (Bn),p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm), and diphenylmethyl(benzhydryl, DPM); silyl groups, such as trimethylsilyl (TMS) andtert-butyldimethylsilyl (TBS); and the like.

General Synthetic Procedures

Compounds of the invention can be prepared from readily availablestarting materials using the following general methods and procedures.Although a particular aspect of the present invention is illustrated inthe schemes below, those skilled in the art will recognize that allaspects of the present invention can be prepared using the methodsdescribed herein or by using other methods, reagents and startingmaterials known to those skilled in the art. It will also be appreciatedthat where typical or preferred process conditions (i.e., reactiontemperatures, times, mole ratios of reactants, solvents, pressures,etc.) are given, other process conditions can also be used unlessotherwise stated. Optimum reaction conditions may vary with theparticular reactants or solvent used, but such conditions can bedetermined by one skilled in the art by routine optimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. The choice of asuitable protecting group for a particular functional group, as well assuitable conditions for protection and deprotection, are well known inthe art. For example, numerous protecting groups, and their introductionand removal, are described in T. W. Greene and G. M. Wuts, ProtectingGroups in Organic Synthesis, Third Edition, Wiley, New York, 1999, andreferences cited therein.

In one method of synthesis, compounds of formulas (I) and (II) areprepared as illustrated in Scheme A. (The substituents and variablesshown in the following schemes have the definitions provided aboveunless otherwise indicated.)

where P¹ represents a hydroxy-protecting group, P² represents ahydroxy-protecting group, and L represents a leaving group, such asbromo. The variable R¹¹ can be hydrogen, or R¹¹ can be P³, where P³represent an amino-protecting group.

As shown in Scheme A, a compound of formula 1 is first reacted with adiamine 2 to provide an intermediate of formula 3. Typically, thisreaction is conducted in a polar, aprotic solvent, optionally in thepresence of base with heating. The protecting group P¹ is typically asilyl protecting group, which is typically removed from the intermediateof formula 3 using a fluoride reagent, for example triethylaminetrihydrofluoride, or an acid, to provide an intermediate of formula 4.The protecting group P² is typically a benzyl protecting group, which istypically removed from the intermediate of formula 4 using a Lewis acidor by hydrogenation using a palladium on carbon catalyst. When R¹¹ is anamino-protecting group P³, the protecting group can be chosen such thatit can be removed under deprotection conditions that are the same asthose for the hydroxy-protecting group P¹. For example, when P¹ isbenzyl, benzyloxycarbonyl (Cbz) can be used as P³ and treatment ofintermediate 4 with a Lewis acid, for example boron trichloride, or byhydrogenation, can provide the product.

The compounds of formula 1 employed in the reactions described in thisapplication are readily prepared by procedures known in the art, anddescribed, for example, in U.S. Pat. Nos. 6,653,323 B2 and 6,670,376 B1,which are incorporated herein by reference, and references therein.

Intermediates of formula 2 can be prepared from readily availablestarting materials. For example, a procedure for the preparation ofintermediate 2′, the intermediate in which R¹ represents anamino-protecting group P³, is illustrated in Scheme B.

In Scheme B, P⁴ represents an amino-protecting group, for example, atert-butoxycarbonyl (Boc) group.

Protected diamine 5 is coupled with a an aldehyde 6, typically in thepresence of a reducing agent, such as sodium cyanoborohydride, toprovide an intermediate of formula 7. Protecting group P³ is added tothe internal nitrogen atom of intermediate 7 to form intermediate 8.Protecting group P³ is chosen such that protecting group P⁴ can beselectively removed in the presence of P³. For example, when Cbz is usedas P³, intermediate 7 is treated with benzylchloroformate, typically inthe presence of base, such as sodium hydroxide, to form intermediate 8,from which protecting group P⁴ is removed, for example, under acidicconditions, to form intermediate 2′.

A procedure for the preparation of intermediate 2″, the intermediate inwhich R¹¹ represents hydrogen, is illustrated in Scheme C.

According to Scheme C, protected-amino-substituted carboxylic acid 9 iscoupled with amine 10 to provide the amide intermediate 11. The reactionis typically conducted in the presence of a coupling agent, for example,1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC), and may employ acatalyst, for example, 1-hydroxybenzotriazole hydrate (HOBT).Intermediate 11 is deprotected under appropriate conditions to providean intermediate of formula 12, which is reduced, for example, using aborane reductant or lithium aluminum hydride, to form intermediate 2″.

Further details regarding specific reaction condition and otherprocedures for preparing representative compounds of the invention orintermediate thereto are described in the Examples below.

Accordingly, in a method aspect, the invention provides a process forpreparing a compound of formula (I), or a salt or stereoisomer orprotected derivative thereof, the process comprising:

reacting a compound of formula (III):

wherein P¹ is a hydroxy-protecting group, L is a leaving group, R⁶ isdefined as in formula (I), each of R^(1a), R^(2a), R^(3a), and R^(4a) isdefined to be the same as R¹, R², R³, and R⁴ in formula (I), or each ofR^(1a), R^(2a), R^(3a), and R^(4a) is independently —OP², wherein P² isa hydroxy-protecting group, with a compound of formula (IV):

wherein R⁵, R⁷, R⁸, R⁹, R¹⁰, n, and in are defined as in formula (I) andR¹¹ is hydrogen or P³ wherein P³ is an amino-protecting group, toprovide a compound of formula (V):

removing the protecting group P¹ to provide a compound of formula (VI):

when any of R^(1a), R^(2a), R^(3a), or R^(4a) is —OP² or when R¹¹ is P³,removing the protecting groups P² and P³, if present, to provide acompound of formula (I), or a salt or stereoisomer thereof.

Pharmaceutical Compositions

The invention also provides pharmaceutical compositions comprising acompound of the invention and a pharmaceutically-acceptable carrier.Accordingly, the compound, preferably in the form of apharmaceutically-acceptable salt, can be formulated for any suitableform of administration, such as oral or parenteral administration, oradministration by inhalation.

By way of illustration, the compound can be admixed with conventionalpharmaceutical carriers and excipients and used in the form of powders,tablets, capsules, elixirs, suspensions, syrups, wafers, and the like.Such pharmaceutical compositions will contain from about 0.05 to about90% by weight of the active compound, and more generally from about 0.1to about 30%. The pharmaceutical compositions may contain commoncarriers and excipients, such as cornstarch or gelatin, lactose,magnesium sulfate, magnesium stearate, sucrose, microcrystallinecellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride, andalginic acid. Disintegrators commonly used in the formulations of thisinvention include croscarmellose, microcrystalline cellulose,cornstarch, sodium starch glycolate and alginic acid.

A liquid composition will generally consist of a suspension or solutionof the compound or pharmaceutically-acceptable salt in a suitable liquidcarrier(s), for example ethanol, glycerine, sorbitol, non-aqueoussolvent such as polyethylene glycol, oils or water, optionally with asuspending agent, a solubilizing agent (such as a cyclodextrin),preservative, surfactant, wetting agent, flavoring or coloring agent.Alternatively, a liquid formulation can be prepared from areconstitutable powder.

For example a powder containing active compound, suspending agent,sucrose and a sweetener can be reconstituted with water to form asuspension; a syrup can be prepared from a powder containing activeingredient, sucrose and a sweetener.

A composition in the form of a tablet can be prepared using any suitablepharmaceutical carrier(s) routinely used for preparing solidcompositions. Examples of such carriers include magnesium stearate,starch, lactose, sucrose, microcrystalline cellulose and binders, forexample polyvinylpyrrolidone. The tablet can also be provided with acolor film coating, or color included as part of the carrier(s). Inaddition, active compound can be formulated in a controlled releasedosage form as a tablet comprising a hydrophilic or hydrophobic matrix.

A composition in the form of a capsule can be prepared using routineencapsulation procedures, for example by incorporation of activecompound and excipients into a hard gelatin capsule. Alternatively, asemi-solid matrix of active compound and high molecular weightpolyethylene glycol can be prepared and filled into a hard gelatincapsule; or a solution of active compound in polyethylene glycol or asuspension in edible oil, for example liquid paraffin or fractionatedcoconut oil can be prepared and filled into a soft gelatin capsule.

Tablet binders that can be included are acacia, methylcellulose, sodiumcarboxymethylcellulose, poly-vinylpyrrolidone (Povidone), hydroxypropylmethylcellulose, sucrose, starch and ethylcellulose. Lubricants that canbe used include magnesium stearate or other metallic stearates, stearicacid, silicone fluid, talc, waxes, oils and colloidal silica.

Flavoring agents such as peppermint, oil of wintergreen, cherryflavoring or the like can also be used. Additionally, it may bedesirable to add a coloring agent to make the dosage form moreattractive in appearance or to help identify the product.

The compounds of the invention and their pharmaceutically-acceptablesalts that are active when given parenterally can be formulated forintramuscular, intrathecal, or intravenous administration.

A typical composition for intramuscular or intrathecal administrationwill consist of a suspension or solution of active ingredient in an oil,for example arachis oil or sesame oil. A typical composition forintravenous or intrathecal administration will consist of a sterileisotonic aqueous solution containing, for example active ingredient anddextrose or sodium chloride, or a mixture of dextrose and sodiumchloride. Other examples are lactated Ringer's injection, lactatedRinger's plus dextrose injection, Normosol-M and dextrose, Isolyte E,acylated Ringer's injection, and the like. Optionally, a co-solvent, forexample, polyethylene glycol; a chelating agent, for example,ethylenediamine tetraacetic acid; a solubilizing agent, for example, acyclodextrin; and an anti-oxidant, for example, sodium metabisulphite,may be included in the formulation. Alternatively, the solution can befreeze dried and then reconstituted with a suitable solvent just priorto administration.

The compounds of this invention and their pharmaceutically-acceptablesalts which are active on topical administration can be formulated astransdermal compositions or transdermal delivery devices (“patches”).Such compositions include, for example, a backing, active compoundreservoir, a control membrane, liner and contact adhesive. Suchtransdermal patches may be used to provide continuous or discontinuousinfusion of the compounds of the present invention in controlledamounts. The construction and use of transdermal patches for thedelivery of pharmaceutical agents is well known in the art. See, forexample, U.S. Pat. No. 5,023,252. Such patches may be constructed forcontinuous, pulsatile, or on demand delivery of pharmaceutical agents.

One preferred manner for administering a compound of the invention isinhalation. Inhalation is an effective means for delivering an agentdirectly to the respiratory tract. There are three general types ofpharmaceutical inhalation devices: nebulizer inhalers, dry powderinhalers (DPI), and metered-dose inhalers (MDI). Conventional nebulizerdevices produce a stream of high velocity air that causes a therapeuticagent to spray as a mist which is carried into the patient's respiratorytract. The therapeutic agent is formulated in a liquid form such as asolution or a suspension of micronized particles of respirable size,where micronized is typically defined as having about 90% or more of theparticles with a diameter of less than about 10 μm.

A typical formulation for use in a conventional nebulizer device is anisotonic aqueous solution of a pharmaceutical salt of the active agentat a concentration of the active agent of between about 0.05 μg/mL andabout 1 mg/mL. Suitable nebulizer devices are provided commercially, forexample, by PARI GmbH (Starnberg, Germany). Other nebulizer devices havebeen disclosed, for example, in U.S. Pat. No. 6,123,068.

DPI's typically administer a therapeutic agent in the form of a freeflowing powder that can be dispersed in a patient's air-stream duringinspiration. Alternative DPI devices which use an external energy sourceto disperse the powder are also being developed. In order to achieve afree flowing powder, the therapeutic agent can be formulated with asuitable excipient (e.g., lactose or starch). A dry powder formulationcan be made, for example, by combining dry lactose particles withmicronized particles of a suitable form, typically apharmaceutically-acceptable salt, of a compound of the invention (i.e.the active agent) and dry blending. Alternatively, the agent can beformulated without excipients. The formulation is loaded into a drypowder dispenser, or into inhalation cartridges or capsules for use witha dry powder delivery device.

Examples of DPI delivery devices provided commercially include Diskhaler(GlaxoSmithKline, Research Triangle Park, N.C.) (see, e.g., U.S. Pat.No. 5,035,237); Diskus (GlaxoSmithKline) (see, e.g., U.S. Pat. No.6,378,519; Turbuhaler (AstraZeneca, Wilmington, Del.) (see, e.g., U.S.Pat. No. 4,524,769); and Rotahaler (GlaxoSmithKline) (see, e.g., U.S.Pat. No. 4,353,365). Further examples of suitable DPI devices aredescribed in U.S. Pat. Nos. 5,415,162, 5,239,993, and 5,715,810 andreferences therein.

MDI's typically discharge a measured amount of therapeutic agent usingcompressed propellant gas. Formulations for MDI administration include asolution or suspension of active ingredient in a liquefied propellant.While chlorofluorocarbons, such as CCl₃F, conventionally have been usedas propellants, due to concerns regarding adverse affects of such agentson the ozone layer, formulations using hydrofluoroalklanes (HFA), suchas 1,1,1,2-tetrafluoroethane (HFA 134a) and1,1,1,2,3,3,3,-heptafluoro-n-propane, (HFA 227) have been developed.Additional components of HFA formulations for MDI administration includeco-solvents, such as ethanol or pentane, and surfactants, such assorbitan trioleate, oleic acid, lecithin, and glycerin. (See, forexample, U.S. Pat. No. 5,225,183, EP 0717987 A2, and WO 92/22286.)

Thus, a suitable formulation for MDI administration can include fromabout 0.001% to about 2% by weight of a compound of the invention, fromabout 0% to about 20% by weight ethanol, and from about 0% to about 5%by weight surfactant, with the remainder being the HFA propellant. Inone approach, to prepare the formulation, chilled or pressurizedhydrofluoroalkane is added to a vial containing the present crystallineform, ethanol (if present) and the surfactant (if present). To prepare asuspension, the compound, typically in salt form, is provided asmicronized particles. The formulation is loaded into an aerosolcanister, which forms a portion of an MDI device. Examples of MDIdevices developed specifically for use with HFA propellants are providedin U.S. Pat. Nos. 6,006,745 and 6,143,227.

In an alternative preparation, a suspension formulation is prepared byspray drying a coating of surfactant on micronized particles of apharmaceutical salt of active compound. (See, for example, WO 99/53901and WO 00/61108.) For additional examples of processes of preparingrespirable particles, and formulations and devices suitable forinhalation dosing see U.S. Pat. Nos. 6,268,533, 5,983,956, 5,874,063,and 6,221,398, and WO 99/55319 and WO 00/30614.

It will be understood that any form of the compounds of the invention,(i.e. free base, pharmaceutical salt, or solvate) that is suitable forthe particular mode of administration, can be used in the pharmaceuticalcompositions discussed above.

The active compounds are expected to be effective over a wide dosagerange and to be administered in a therapeutically effective amount. Itwill be understood, however, that the amount of the compound actuallyadministered will be determined by a physician, in the light of therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered and itsrelative activity, the age, weight, and response of the individualpatient, the severity of the patient's symptoms, and the like.

A compound can be administered in a periodic dose: weekly, multipletimes per week, daily, or multiple doses per day. The treatment regimenmay require administration over extended periods of time, for example,for several weeks or months, or the treatment regimen may requirechronic administration.

Suitable doses of the therapeutic agents for inhalation administrationare in the general range of from about 0.05 μg/day to about 1000 μg/day,including from about 0.1 μg/day to about 500 μg/day. It will beunderstood that the fraction of active agent delivered to the lungcharacteristic of particular delivery devices is taken into account indetermining suitable doses for inhalation administration. Suitable dosesfor oral administration are in the general range of from about 0.05μg/day to about 100 mg/day, including from about 0.5 to about 1000μg/day.

Among other properties, compounds of the invention exhibit surprisingand unexpected duration of action. As described in the examples below, acompound of the invention demonstrated duration of action greater than24 hours in an animal model of bronchoprotection. Furthermore compoundsof the invention are potent and selective agonists of the β₂ adrenergicreceptor. In particular, compounds of the invention are selective forthe β₂ adrenergic receptor as compared with the β₁ and β₃ adrenergicreceptors.

The invention thus provides a method of treating a mammal having adisease or condition associated with β₂ adrenergic receptor activity,the method comprising administering to the mammal a therapeuticallyeffective amount of a compound of the invention or of a pharmaceuticalcomposition comprising a compound of the invention.

The present active agents can also used as part of a combinationcomprising, in addition, one or more other therapeutic agents. Forexample, the present agents can be administered together with one ormore therapeutic agents selected from anti-inflammatory agents (e.g.corticosteroids and non-steroidal anti-inflammatory agents (NSAIDs),antichlolinergic agents (particularly muscarinic receptor antagonists),other β₂ adrenergic receptor agonists, antiinfectives agents (e.g.antibiotics or antivirals) or antihistamines. The invention thusprovides, in a further aspect, a combination comprising a compound ofthe invention and one or more therapeutic agents, for example, ananti-inflammatory agent, an antichlolinergic agent, another β₂adrenergic receptor agonist, an antiinfectives agent or anantihistamine.

The other therapeutic agents can be used in the form ofpharmaceutically-acceptable salts or solvates. As appropriate, the othertherapeutic agents can be used as optically pure stereoisomers.

Suitable anti-inflammatory agents include corticosteroids and NSAIDs.Suitable corticosteroids which may be used in combination with thecompounds of the invention are those oral and inhaled corticosteroidsand their pro-drugs which have anti-inflammatory activity. Examplesinclude methyl prednisolone, prednisolone, dexamethasone, fluticasonepropionate,6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester,6α,9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-propionyloxy-androsta-1,4-diene-17β-carbothioicacid S-(2-oxo-tetrahydro-furan-3S-yl) ester, beclomethasone esters (e.g.the 17-propionate ester or the 17,21-dipropionate ester), budesonide,flunisolide, mometasone esters (e.g. the furoate ester), triamcinoloneacetonide, rofleponide, ciclesonide, butixocort propionate, RPR-106541,and ST-126. Preferred corticosteroids include fluticasone propionate,6α,9α-difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl-1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester and6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester, more preferably6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester.

Suitable NSAIDs include sodium cromoglycate; nedocromil sodium;phosphodiesterase (PDE) inhibitors (e.g. theophylline, PDE4 inhibitorsor mixed PDE3/PDE4 inhibitors); leukotriene antagonists (e.g.monteleukast); inhibitors of leukotriene synthesis; iNOS inhibitors;protease inhibitors, such as tryptase and elastase inhibitors; beta-2integrin antagonists and adenosine receptor agonists or antagonists(e.g. adenosine 2a agonists); cytokine antagonists (e.g. chemokineantagonists such as, an interleukin antibody (αIL antibody),specifically, an αIL-4 therapy, an αIL-13 therapy, or a combinationthereof); or inhibitors of cytokine synthesis. Suitable otherβ₂-adrenoreceptor agonists include salmeterol (e.g. as the xinafoate),salbutamol (e.g. as the sulphate or the free base), formoterol (e.g. asthe fumarate), fenoterol or terbutaline and salts thereof.

Also of interest is use of the present active agent in combination witha phosphodiesterase 4 (PDE4) inhibitor or a mixed PDE3/PDE4 inhibitor.Representative phosphodiesterase-4 (PDE4) inhibitors or mixed PDE3/PDE4inhibitors include, but are not limited to cis4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)cyclohexan-1-carboxylicacid,2-carbomethoxy-4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-one;cis-[4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-ol];cis-4-cyano-4-[3-(cyclopentyloxy)-4-methoxyphenyl]cyclohexane-1-carboxylicacid and the like, or pharmaceutically-acceptable salts thereof. Otherrepresentative PDE4 or mixed PDE4/PDE3 inhibitors include AWD-12-281(elbion); NCS-613 (INSERM); D-4418 (Chiroscience and Schering-Plough);CI-1018 or PD-168787 (Pfizer); benzodioxole compounds disclosed inWO99/16766 (Kyowa Hakko); K-34 (Kyowa Hakko); V-11294A (Napp);roflumilast (Byk-Gulden); pthalazinone compounds disclosed in WO99/47505(Byk-Gulden); Pumafentrine (Byk-Gulden, now Altana); arofylline(Almirall-Prodesfarma); VM554/UM565 (Vernalis); T-440 (Tanabe Seiyaku);and T2585 (Tanabe Seiyaku).

Suitable anticholinergic agents are those compounds that act asantagonists at the muscarinic receptor, in particular those compoundswhich are antagonists of the M₁, M₂, or M₃ receptors, or of combinationsthereof. Exemplary compounds include the alkaloids of the belladonnaplants as illustrated by the likes of atropine, scopolamine,homatropine, hyoscyamine; these compounds are normally administered as asalt, being tertiary amines. These drugs, particularly the salt forms,are readily available from a number of commercial sources or can be madeor prepared from literature data via, to wit:

Atropine—CAS-51-55-8 or CAS-51-48-1 (anhydrous form), atropine sulfate—CAS-5908-99-6; atropine oxide—CAS-4438-22-6 or its HClsalt—CAS-4574-60-1 and methylatropine nitrate —CAS-52-88-0.

Homatropine—CAS-87-00-3, hydrobromide salt —CAS-51-56-9, methylbromidesalt —CAS-80-49-9.

Hyoscyamine (d, l)—CAS-101-31-5, hydrobromide salt —CAS-306-03-6 andsulfate salt—CAS-6835-16-1.

Scopolamine—CAS-51-34-3, hydrobromide salt—CAS-6533-68-2, methylbromidesalt-CAS-155-41-9.

Preferred anticholinergics include ipratropium (e.g. as the bromide),sold under the name Atrovent, oxitropium (e.g. as the bromide) andtiotropium (e.g. as the bromide) (CAS-139404-48-1). Also of interestare: methantheline (CAS-53-46-3), propantheline bromide (CAS-50-34-9),anisotropine methyl bromide or Valpin 50 (CAS-80-50-2), clidiniumbromide (Quarzan, CAS-3485-62-9), copyrrolate (Robinul), isopropamideiodide (CAS-71-81-8), mepenzolate bromide (U.S. Pat. No. 2,918,408),tridihexethyl chloride (Pathilone, CAS-4310-35-4), and hexocycliummethylsulfate (Tral, CAS-115-63-9). See also cyclopentolatehydrochloride (CAS-5870-29-1), tropicamide (CAS-1508-75-4),trihexyphenidyl hydrochloride (CAS-144-11-6), pirenzepine(CAS-29868-97-1), telenzepine (CAS-80880-90-9), AF-DX 116, ormethoctramine, and the compounds disclosed in WO01/04118, the disclosureof which is hereby incorporated by reference.

Suitable antihistamines (also referred to as H₁-receptor antagonists)include any one or more of the numerous antagonists known which inhibitH₁-receptors, and are safe for human use. All are reversible,competitive inhibitors of the interaction of histamine withH₁-receptors. The majority of these inhibitors, mostly first generationantagonists, are characterized, based on their core structures, asethanolamines, ethylenediamines, and alkylamines. In addition, otherfirst generation antihistamines include those which can be characterizedas based on piperizine and phenothiazines. Second generationantagonists, which are non-sedating, have a similar structure-activityrelationship in that they retain the core ethylene group (thealkylamines) or mimic a tertiary amine group with piperizine orpiperidine. Exemplary antagonists are as follows:

Ethanolamines: carbinoxamine maleate, clemastine fumarate,diphenylhydramine hydrochloride, and dimenhydrinate.

Ethylenediamines: pyrilamine amleate, tripelennamine HCl, andtripelennamine citrate.

Alkylamines: chlorpheniramine and its salts such as the maleate salt,and acrivastine.

Piperazines: hydroxyzine HCl, hydroxyzine pamoate, cyclizine HCl,cyclizine lactate, meclizine HCl, and cetirizine HCl.

Piperidines: Astemizole, levocabastine HCl, loratadine or itsdescarboethoxy analogue, and terfenadine and fexofenadine hydrochlorideor another pharmaceutically-acceptable salt.

Azelastine hydrochloride is yet another H₁ receptor antagonist which maybe used in combination with a compound of the invention.

Examples of preferred anti-histamines include methapyrilene andloratadine.

The invention thus provides, in a further aspect, a combinationcomprising a compound of formula (I) or a pharmaceutically-acceptablesalt or solvate or stereoisomer thereof and a corticosteroid. Inparticular, the invention provides a combination wherein thecorticosteroid is fluticasone propionate or wherein the corticosteroidis6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester or6α,9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-propionyloxy-androsta-1,4-diene-17β-carbothioicacid S-(2-oxo-tetrahydro-furan-3S-yl) ester.

The invention thus provides, in a further aspect, a combinationcomprising a compound of formula (I) or a pharmaceutically-acceptablesalt or solvate or stereoisomer thereof and a PDE4 inhibitor.

The invention thus provides, in a further aspect, a combinationcomprising a compound of formula (I) or a pharmaceutically-acceptablesalt or solvate or stereoisomer thereof and an anticholinergic agent.

The invention thus provides, in a further aspect, a combinationcomprising a compound of formula (I) or a pharmaceutically-acceptablesalt or solvate or stereoisomer thereof and an antihistamine.

The invention thus provides, in a further aspect, a combinationcomprising a compound of formula (I) or a pharmaceutically-acceptablesalt or solvate or stereoisomer thereof together with a PDE4 inhibitorand a corticosteroid.

The invention thus provides, in a further aspect, a combinationcomprising a compound of formula (I) or a pharmaceutically-acceptablesalt or solvate or stereoisomer thereof together with an anticholinergicagent and a corticosteroid.

As used in the above combinations, the term, “a compound of formula (I)”includes a compound of formula (II) and preferred groups thereof, andany individually disclosed compound or compounds.

Accordingly, the invention further provides pharmaceutical compositionscomprising a compound of formula (I) or a pharmaceutically-acceptablesalt or solvate or stereoisomer and one or more other therapeuticagents, as described above.

The individual compounds of such combinations may be administered eithersequentially or simultaneously in separate or combined pharmaceuticalformulations. Appropriate doses of known therapeutic agents will bereadily appreciated by those skilled in the art. Methods of treatment ofthe invention, therefore, include administration of the individualcompounds of such combinations either sequentially or simultaneously inseparate or combined pharmaceutical formulations.

Thus, according to a further aspect, the invention provides a method oftreating a mammal having a disease or condition associated with β₂adrenergic receptor activity, the method comprising administering to themammal a therapeutically effective amount of a compound of formula (I)or a pharmaceutically-acceptable salt or solvate or stereoisomer and atherapeutically-acceptable amount of one or more other therapeuticagents.

Since compounds of the invention are β₂ adrenergic receptor agonists,such compounds are also useful as research tools for investigating orstudying biological systems or samples having β₂ adrenergic receptors,or for discovering new β₂ adrenergic receptor agonists. Moreover, sincecompounds of the invention exhibit selectivity for β₂ adrenergicreceptors as compared with binding and functional activity at receptorsof other β adrenergic subtypes, such compounds are also useful forstudying the effects of selective agonism of β₂ adrenergic receptors ina biological system or sample. Any suitable biological system or samplehaving β₂ adrenergic receptors may be employed in such studies which maybe conducted either in vitro or in vivo.

Representative biological systems or samples suitable for such studiesinclude, but are not limited to, cells, cellular extracts, plasmamembranes, tissue samples, mammals (such as mice, rats, guinea pigs,rabbits, dogs, pigs, etc.) and the like. The effects of agonizing the β₂adrenergic receptor are determined using conventional procedures andequipment, such as radioligand binding assays and functional assays, forexample the assay for ligand-mediated changes in intracellular cyclicadenosine monophosphate (cAMP) described below, or assays of a similarnature. A β₂ adrenergic receptor-agonizing amount of a compound of theinvention will typically range from about 1 nanomolar to about 1000nanomolar. When compounds of the invention are used as research toolsfor discovering new β₂ adrenergic receptor agonists, the invention alsoincludes, as separate embodiments, both the generation of comparisondata (using the appropriate assays) and the analysis of the test data toidentify test compounds of interest.

The following non-limiting examples illustrate representativepharmaceutical compositions of the invention. Additional suitablecarriers for formulations of the active compounds of the presentinvention can also be found in Remington: The Science and Practice ofPharmacy, 20th Edition, Lippincott Williams & Wilkins, Philadelphia,Pa., 2000.

FORMULATION EXAMPLE A

This example illustrates the preparation of a representativepharmaceutical composition for oral administration of a compound of thisinvention: Ingredients Quantity per tablet, (mg) Active Ingredient 1Lactose, spray-dried 148 Magnesium stearate 2

The above ingredients are mixed and introduced into a hard-shell gelatincapsule.

FORMULATION EXAMPLE B

This example illustrates the preparation of another representativepharmaceutical composition for oral administration of a compound of thisinvention: Ingredients Quantity per tablet, (mg) Active Ingredient 1Cornstarch 50 Lactose 145 Magnesium stearate 5

The above ingredients are mixed intimately and pressed into singlescored tablets.

FORMULATION EXAMPLE C

This example illustrates the preparation of a representativepharmaceutical composition for oral administration of a compound of thisinvention.

An oral suspension is prepared having the following composition.Ingredients Active Compound 3 mg Fumaric acid 0.5 g Sodium chloride 2.0g Methyl paraben 0.1 g Granulated sugar 25.5 g Sorbitol (70% solution)12.85 g Veegum K (Vanderbilt Co.) 1.0 g Flavoring 0.035 mL Colorings 0.5mg Distilled water q.s. to 100 mL

FORMULATION EXAMPLE D

This example illustrates the preparation of a representativepharmaceutical composition containing a compound of this invention.

An injectable preparation buffered to a pH of 4 is prepared having thefollowing composition: Ingredients Active Compound 0.1 mg Sodium AcetateBuffer Solution (0.4 M) 2.0 mL HCl (1N) q.s. to pH 4 Water (distilled,sterile) q.s. to 20 mL

FORMULATION EXAMPLE E

This example illustrates the preparation of a representativepharmaceutical composition for injection of a compound of thisinvention.

A reconstituted solution is prepared by adding 20 mL of sterile water to1 mg of the compound of this invention. Before use, the solution is thendiluted with 200 mL of an intravenous fluid that is compatible with theactive compound. Such fluids are chosen from 5% dextrose solution, 0.9%sodium chloride, or a mixture of 5% dextrose and 0.9% sodium chloride.Other examples are lactated Ringer's injection, lactated Ringer's plus5% dextrose injection, Normosol-M and 5% dextrose, Isolyte E, andacylated Ringer's injection.

FORMULATION EXAMPLE F

This example illustrates the preparation of a representativepharmaceutical composition for topical application of a compound of thisinvention. Ingredients grams Active compound 0.2-10 Span 60 2 Tween 60 2Mineral oil 5 Petrolatum 10 Methyl paraben 0.15 Propyl paraben 0.05 BHA(butylated hydroxy anisole) 0.01 Water q.s. to 100

All of the above ingredients, except water, are combined and heated to60° C. with stirring. A sufficient quantity of water at 60° C. is thenadded with vigorous stirring to emulsify the ingredients, and water thenadded q.s. 100 g.

FORMULATION EXAMPLE G

This example illustrates the preparation of a representativepharmaceutical composition containing a compound of the invention.

An aqueous aerosol formulation for use in a nebulizer is prepared bydissolving 0.1 mg of a pharmaceutical salt of active compound in a 0.9%sodium chloride solution acidified with citric acid. The mixture isstirred and sonicated until the active salt is dissolved. The pH of thesolution is adjusted to a value in the range of from 3 to 8 by the slowaddition of NaOH.

FORMULATION EXAMPLE H

This example illustrates the preparation of a dry powder formulationcontaining a compound of the invention for use in inhalation cartridges.

Gelatin inhalation cartridges are filled with a pharmaceuticalcomposition having the following ingredients: Ingredients mg/cartridgePharmaceutical salt of active compound 0.2 Lactose 25

The pharmaceutical salt of active compound is micronized prior toblending with lactose. The contents of the cartridges are administeredusing a powder inhaler.

FORMULATION EXAMPLE I

This example illustrates the preparation of a dry powder formulationcontaining a compound of the invention for use in a dry powderinhalation device.

A pharmaceutical composition is prepared having a bulk formulation ratioof micronized pharmaceutical salt to lactose of 1:200. The compositionis packed into a dry powder inhalation device capable of deliveringbetween about 10 μg and about 100 μg of active drug ingredient per dose.

FORMULATION EXAMPLE J

This example illustrates the preparation of a formulation containing acompound of the invention for use in a metered dose inhaler.

A suspension containing 5% pharmaceutical salt of active compound, 0.5%lecithin, and 0.5% trehalose is prepared by dispersing 5 g of activecompound as micronized particles with mean size less than 10 μm in acolloidal solution formed from 0.5 g of trehalose and 0.5 g of lecithindissolved in 100 mL of demineralized water. The suspension is spraydried and the resulting material is micronized to particles having amean diameter less than 1.5 μm. The particles are loaded into canisterswith pressurized 1,1,1,2-tetrafluoroethane.

FORMULATION EXAMPLE K

This example illustrates the preparation of a formulation containing acompound of the invention for use in a metered dose inhaler.

A suspension containing 5% pharmaceutical salt of active compound and0.1% lecithin is prepared by dispersing 10 g of active compound asmicronized particles with mean size less than 10 μm in a solution formedfrom 0.2 g of lecithin dissolved in 200 mL of demineralized water. Thesuspension is spray dried and the resulting material is micronized toparticles having a mean diameter less than 1.5 μm. The particles areloaded into canisters with pressurized1,1,1,2,3,3,3-heptafluoro-n-propane.

FORMULATION EXAMPLE L

This example illustrates the preparation of a dry powder formulationcontaining a compound of the invention and a corticosteroid for use ininhalation cartridges.

Gelatin inhalation cartridges are filled with a pharmaceuticalcomposition having the following ingredients: Ingredients mg/cartridgePharmaceutical salt of active compound 0.1 Corticosteroid 0.5 Lactose 25

The pharmaceutical salt of active compound and the corticosteroid aremicronized prior to blending with lactose. The contents of thecartridges are administered using a powder inhaler.

Biological Assays

The compounds of this invention, and their pharmaceutically-acceptablesalts, exhibit biological activity and are useful for medical treatment.The ability of a compound to bind to the β2 adrenergic receptor, as wellas its selectivity, agonist potency, and intrinsic activity can bedemonstrated using Tests A-B below, or can be demonstrated using othertests that are known in the art.

Abbreviations

-   -   % Eff % efficacy    -   ATCC American Type Culture Collection    -   BSA Bovine Serum Albumin    -   cAMP Adenosine 3′:5′-cyclic monophosphate DMEM Dulbecco's        Modified Eagle's Medium DMSO Dimethyl sulfoxide    -   EDTA Ethylenediaminetetraacetic acid    -   Emax maximal efficacy    -   FBS Fetal bovine serum    -   Gly Glycine    -   HEK-293 Human embryonic kidney-293    -   PBS Phosphate buffered saline    -   rpm rotations per minute    -   Tris Tris(hydroxymethyl)aminomethane

Membrane Preparation from Cells Expressing Human β₁ or β₂ AdrenergicReceptors

HEK-293 derived cell lines stably expressing cloned human β₁ or β₂adrenergic receptors, respectively, were grown to near confluency inDMEM with 10% dialyzed FBS in the presence of 500 μg/mL Geneticin. Thecell monolayer was lifted with Versene 1:5,000 (0.2 g/L EDTA in PBS)using a cell scraper. Cells were pelleted by centrifugation at 1,000rpm, and cell pellets were either stored frozen at −80° C. or membraneswere prepared immediately. For preparation, cell pellets wereresuspended in lysis buffer (10 mM Tris/HCL pH 7.4 @ 4° C., one tabletof “Complete Protease Inhibitor Cocktail Tablets with 2 mM EDTA” per 50mL buffer (Roche cat.# 1697498, Roche Molecular Biochemicals,Indianapolis, Ind.)) and homogenized using a tight-fitting Dounce glasshomogenizer (20 strokes) on ice. The homogenate was centrifuged at20,000×g, the pellet was washed once with lysis buffer by resuspensionand centrifugation as above. The final pellet was resuspended inmembrane buffer (75 mM Tris/HCl pH 7.4, 12.5 nM MgCl₂, 1 mM EDTA @ 25°C.). Protein concentration of the membrane suspension was determined bythe method of Bradford (Bradford M M., Analytical Biochemistry, 1976,72, 248-54). Membranes were stored frozen in aliquots at −80° C.

Test A Radioligand Binding Assay on Human β₁ and β₂ Adrenergic Receptors

Binding assays were performed in 96-well microtiter plates in a totalassay volume of 100 μL with 5 μg membrane protein for membranescontaining the human β₂ adrenergic receptor, or 2.5 μg membrane proteinfor membranes containing the human β₁ adrenergic receptor in assaybuffer (75 mM Tris/HCl pH 7.4 @ 25° C., 12.5 mM MgCl₂, 1 mM EDTA, 0.2%BSA). Saturation binding studies for determination of K_(d) values ofthe radioligand were done using [³H]dihydroalprenolol (NET-720, 100Ci/mmol, PerkinElmer Life Sciences Inc., Boston, Mass.) at 10 differentconcentrations ranging from 0.01 nM-200 nM. Displacement assays fordetermination of pK_(i) values of compounds were done with[³H]dihydroalprenolol at 1 nM and 10 different concentrations ofcompound ranging from 40 pM-10 μM. Compounds were dissolved to aconcentration of 10 mM in dissolving buffer (25 mM Gly-HCl pH 3.0 with50% DMSO), then diluted to 1 mM in 50 mM Gly-HCl pH 3.0, and from thereserially diluted into assay buffer. Non-specific binding was determinedin the presence of 10 μM unlabeled alprenolol. Assays were incubated for90 minutes at room temperature, binding reactions were terminated byrapid filtration over GF/B glass fiber filter plates (Packard BioScienceCo., Meriden, Conn.) presoaked in 0.3% polyethyleneimine. Filter plateswere washed three times with filtration buffer (75 mM Tris/HCl pH 7.4 @4° C., 12.5 mM MgCl₂, 1 mM EDTA) to remove unbound radioactivity. Plateswere dried, 50 μL Microscint-20 liquid scintillation fluid (PackardBioScience Co., Meriden, Conn.) was added and plates were counted in aPackard Topcount liquid scintillation counter (Packard BioScience Co.,Meriden, Conn.). Binding data were analyzed by nonlinear regressionanalysis with the GraphPad Prism Software package (GraphPad Software,Inc., San Diego, Calif.) using the 3-parameter model for one-sitecompetition. The curve minimum was fixed to the value for nonspecificbinding, as determined in the presence of 10 μM alprenolol. K_(i) valuesfor compounds were calculated from observed IC₅₀ values and the K_(d)value of the radioligand using the Cheng-Prusoff equation (Cheng Y, andPrusoff W H., Biochemical Pharmacology, 1973, 22, 23, 3099-108). Thereceptor subtype selectivity was calculated as the ratio ofK_(i)(β₁)/K_(i)(β₂).

Binding results are expressed as the negative decadic logarithm of theK_(i) values, pK_(i). Compounds having a higher pK_(i) value in thisassay have a higher binding affinity for the β₂ adrenergic receptor.

Test B Whole-Cell cAMP Flashplate Assays with Cell Lines HeterologouslyExpressing Human β₁ Adrenoceptor, β₂ Adrenoceptor, and β₃ Adrenoceptor,Respectively

A HEK-293 cell line stably expressing cloned human β₁ adrenergicreceptor (clone H34.1) was grown to about 70%-90% confluency in mediumconsisting of DMEM supplemented with 10% FBS and 500 μg/mL Geneticin. AHEK-293 cell line stably expressing cloned human β₂-adrenoceptor (cloneH24.14) was grown in the same medium to full confluency. A CHO-K1 cellline stably expressing cloned human β₃-adrenoceptor was grown to about70%-90% confluency in Ham's F-12 medium supplemented with 10% FBS andwith 800 μg/mL Geneticin added to every fifth passage. The day beforethe assay, cultures were switched to the same growth-media withoutantibiotics.

cAMP assays were performed in a radioimmunoassay format using theFlashplate Adenylyl Cyclase Activation Assay System with ¹²⁵I-cAMP (NENSMP004, PerkinElmer Life Sciences Inc., Boston, Mass.), according to themanufacturers instructions.

On the day of the assay, cells were rinsed once with PBS, lifted withVersene 1:5,000 (0.2 g/L EDTA in PBS) and counted. Cells were pelletedby centrifugation at 1,000 rpm and resuspended in stimulation bufferprewarmed to 37° C. For cells expressing the β₁-adrenoceptor, 10 nM ICI118,551 were added to the stimulation buffer, and cells were incubatedfor 10 min at 37° C. Cells were used at final concentrations of 30,000,40,000 and 70,000 cells/well for the β₁-adrenoceptor-, theβ₂-adrenoceptor- and the β₃-adrenoceptor expressing cells, respectively.Compounds were dissolved to a concentration of 10 mM in DMSO, thendiluted to 1 mM in 50 mM Gly-HCl pH 3.0, and from there serially dilutedinto assay buffer (75 mM Tris/HCl pH 7.4 @ 25° C., 12.5 mM MgCl₂, 1 mMEDTA, 0.2% BSA). Compounds were tested in the assay at 11 differentconcentrations, ranging from 10 μM to 9.5 μM. Reactions were incubatedfor 10 min at 37° C. and stopped by addition of 100 μL ice-colddetection buffer. Plates were sealed, incubated over night at 4° C. andcounted the next morning in a topcount scintillation counter (PackardBioScience Co., Meriden, Conn.). The amount of cAMP produced per mL ofreaction was calculated based on the counts observed for the samples andcAMP standards, as described in the manufacturer's user manual. Datawere analyzed by nonlinear regression analysis with the GraphPad PrismSoftware package (GraphPad Software, Inc., San Diego, Calif.) using the3-parameter model for sigmoidal dose-response (Hill slope=1). Agonistpotencies were expressed as pEC₅₀ values. Functional β₂/β₁ selectivitywas defined as the ratio EC₅₀(β₁)/EC₅₀(β₂), and correspondinglyfunctional β₂/β₃ selectivity was defined as the ratio EC₅₀(β₃)/EC₅₀(β₂).

Test C Whole-Cell Camp Flashplate Assay with a Lung Epithelial Cell LineEndogenously Expressing Human β₂ Adrenergic Receptor

For the determination of agonist potencies and efficacies (intrinsicactivities) in a cell line expressing endogenous levels of β2 adrenergicreceptor, a human lung epithelial cell line (BEAS-2B) was used (ATCCCRL-9609, American Type Culture Collection, Manassas, Va.) (January B,et al., British Journal of Pharmacology, 1998, 123, 4, 701-11). Cellswere grown to 75-90% confluency in complete, serum-free medium (LHC-9MEDIUM containing Epinephrine and Retinoic Acid, cat #181-500, BiosourceInternational, Camarillo, Calif.). The day before the assay, medium wasswitched to LHC-8 (No epinephrine or retinoic acid, cat #141-500,Biosource International, Camarillo, Calif.).

cAMP assays were performed in a radioimmunoassay format using theFlashplate Adenylyl Cyclase Activation Assay System with ¹²⁵I-cAMP (NENSMP004, PerkinElmer Life Sciences Inc., Boston, Mass.), according to themanufacturers instructions.

On the day of the assay, cells were rinsed with PBS, lifted by scrapingwith 5 mM EDTA in PBS, and counted. Cells were pelleted bycentrifugation at 1,000 rpm and resuspended in stimulation bufferprewarmed to 37° C. at a final concentration of 600,000 cells/mL. Cellswere used at a final concentration of 30,000 cells/well in the assay.Compounds were dissolved to a concentration of 10 mM in dissolvingbuffer (25 mM Gly-HCl pH 3.0 with 50% DMSO), then diluted to 1 mM in 50mM Gly-HCl pH 3.0, and from there serially diluted into assay buffer (75mM Tris/HCl pH 7.4 @ 25° C., 12.5 mM MgCl₂, 1 mM EDTA, 0.2% BSA).

Compounds were tested in the assay at 10 different concentrations,ranging from 10 μM to 40 μM. Maximal response was determined in thepresence of 10 μM Isoproterenol. Reactions were incubated for 10 min at37° C. and stopped by addition of 100 μl ice-cold detection buffer.Plates were sealed, incubated over night at 4° C. and counted the nextmorning in a topcount scintillation counter (Packard BioScience Co.,Meriden, Conn.). The amount of cAMP produced per mL of reaction wascalculated based on the counts observed for samples and cAMP standards,as described in the manufacturer's user manual. Data were analyzed bynonlinear regression analysis with the GraphPad Prism Software package(GraphPad Software, Inc., San Diego, Calif.) using the 4-parameter modelfor sigmoidal dose-response with variable slope. Compound efficacy (%Eff) was calculated from the ratio of the observed Emax (TOP of thefitted curve) and the maximal response obtained for 10 μM isoproterenoland was expressed as % Eff relative to isoproterenol.

Test D Assay Of Bronchoprotection Against Acetylcholine-InducedBronchospasm in a Guinea Pig Model

Groups of 6 male guinea pigs (Duncan-Hartley (HsdPoc:DH) Harlan,Madison, Wis.) weighing between 250 and 350 g were individuallyidentified by cage cards. Throughout the study animals were allowedaccess to food and water ad libitum.

Test compounds were administered via inhalation over 10 minutes in awhole-body exposure dosing chamber (R&S Molds, San Carlos, Calif.). Thedosing chambers were arranged so that an aerosol was simultaneouslydelivered to 6 individual chambers from a central manifold. Following a60 minute acclimation period and a 10 minute exposure to nebulized waterfor injection (WFI), guinea pigs were exposed to an aerosol of testcompound or vehicle (WFI). These aerosols were generated from aqueoussolutions using an LC Star Nebulizer Set (Model 22F51, PARI RespiratoryEquipment, Inc. Midlothian, Va.) driven by a mixture of gases (CO₂=5%,O₂=21% and N₂=74%) at a pressure of 22 psi. The gas flow through thenebulizer at this operating pressure was approximately 3 L/minute. Thegenerated aerosols were driven into the chambers by positive pressure.No dilution air was used during the delivery of aerosolized solutions.During the 10 minute nebulization, approximately 1.8 mL of solution wasnebulized. This was measured gravimetrically by comparing pre- andpost-nebulization weights of the filled nebulizer.

The bronchoprotective effects of compounds administered via inhalationwere evaluated using whole body plethysmography at 1.5, 24, 48 and 72hours post-dose. Forty-five minutes prior to the start of the pulmonaryevaluation, each guinea pig was anesthetized with an intramuscularinjection of ketamine (43.75 mg/kg), xylazine (3.50 mg/kg) andacepromazine (1.05 mg/kg). After the surgical site was shaved andcleaned with 70% alcohol, a 2-5 cm midline incision of the ventralaspect of the neck was made. Then, the jugular vein was isolated andcannulated with a saline-filled polyethylene catheter (PE-50, BectonDickinson, Sparks, Md.) to allow for intravenous infusions of a 0.1mg/mL solution of acetylcholine (Ach), (Sigma-Aldrich, St. Louis, Mo.)in saline. The trachea was then dissected free and cannulated with a 14Gteflon tube (#NE-014, Small Parts, Miami Lakes, Fla.). If required,anesthesia was maintained by additional intramuscular injections of theaforementioned anesthetic cocktail. The depth of anesthesia wasmonitored and adjusted if the animal responded to pinching of its paw orif the respiration rate was greater than 100 breaths/minute.

Once the cannulations were complete, the animal was placed into aplethysmograph (#PLY3114, Buxco Electronics, Inc., Sharon, Conn.) and anesophageal pressure cannula was inserted to measure pulmonary drivingpressure (pressure). The teflon tracheal tube was attached to theopening of the plethysmograph to allow the guinea pig to breathe roomair from outside the chamber. The chamber was then sealed. A heatinglamp was used to maintain body temperature and the guinea pig's lungswere inflated 3 times with 4 mL of air using a 10 mL calibration syringe(#5520 Series, Hans Rudolph, Kansas City, Mo.) to ensure that the lowerairways had not collapsed and that the animal did not suffer fromhyperventilation.

Once it was determined that baseline values were within the range0.3-0.9 mL/cm H₂O for compliance and within the range 0.1-0.199 cmH₂O/mL per second for resistance, the pulmonary evaluation wasinitiated. A Buxco pulmonary measurement computer program enabled thecollection and derivation of pulmonary values. Starting this programinitiated the experimental protocol and data collection. The changes involume over time that occurred within the plethysmograph with eachbreath were measured via a Buxco pressure transducer. By integratingthis signal over time, a measurement of flow was calculated for eachbreath. This signal, together with the pulmonary driving pressurechanges, which were collected using a Sensym pressure transducer(#TRD4100), was connected via a Buxco (MAX 2270) preamplifier to a datacollection interface (#'s SFT3400 and SFT3813). All other pulmonaryparameters were derived from these two inputs.

Baseline values were collected for 5 minutes, after which time theguinea pigs were challenged with Ach. Ach was infused intravenously for1 minute from a syringe pump (sp210iw, World Precision Instruments,Inc., Sarasota, Fla.) at the following doses and prescribed times fromthe start of the experiment: 1.9 μg/minute at 5 minutes, 3.8 μg/minuteat 10 minutes, 7.5 μg/minute at 15 minutes, 15.0 μg/minute at 20minutes, 30 μg/minute at 25 minutes and 60 μg/minute at 30 minutes. Ifresistance or compliance had not returned to baseline values at 3minutes following each Ach dose, the guinea pig's lungs were inflated 3times with 4 mL of air from a 10 mL calibration syringe. Recordedpulmonary parameters included respiration frequency (breaths/minute),compliance (mL/cm H₂O) and pulmonary resistance (cm H₂O/mL per second)(Giles et al., 1971). Once the pulmonary function measurements werecompleted at minute 35 of this protocol, the guinea pig was removed fromthe plethysmograph and euthanized by CO₂ asphyxiation.

The quantity PD₂, which is defined as the amount of Ach needed to causea doubling of the baseline pulmonary resistance, was calculated usingthe pulmonary resistance values derived from the flow and the pressureover a range of Ach challenges using the following equation. This wasderived from the equation used to calculate PC₂₀ values in the clinic(Am. Thoracic Soc, 2000).${PD}_{2} = {{antilog}\left\lbrack {{\log\quad C_{1}} + \frac{\left( {{\log\quad C_{2}} - {\log\quad C_{1}}} \right)\left( {{2\quad R_{0}} - R_{1}} \right)}{R_{2} - R_{1}}} \right\rbrack}$

where:

C₁=Second to last Ach concentration (concentration preceding C₂)

C₂=Final concentration of Ach (concentration resulting in a 2-foldincrease in pulmonary resistance (R_(L)))

R₀=Baseline R_(L) value

R₁=R_(L) value after C₁

R₂=R_(L) value after C₂

Statistical analysis of the data was performed using a One-Way Analysisof Variance followed by post-hoc analysis using a Bonferroni/Dunn test.A P-value <0.05 was considered significant.

Dose-response curves were fitted with a four parameter logistic equationusing GraphPad Prism, version 3.00 for Windows (GraphPad Software, SanDiego, Calif.)Y=Min+(Max−Min)/(1+10ˆ((log ED ₅₀ −X)*Hillslope)),

where X is the logarithm of dose, Y is the response (PD₂), and Y startsat Min and approaches asymptotically to Max with a sigmoidal shape.

The following examples are offered to illustrate the invention, and arenot to be construed in any way as limiting the scope of the invention.

EXAMPLES

General: Unless noted otherwise, reagents, starting material andsolvents were purchased from commercial suppliers, for exampleSigma-Aldrich (St. Louis, Mo.), J. T. Baker (Phillipsburg, N.J.), andHoneywell Burdick and Jackson (Muskegon, Mich.), and used withoutfurther purification; reactions were run under nitrogen atmosphere;reaction mixtures were monitored by thin layer chromatography (silicaTLC), analytical high performance liquid chromatography (anal. HPLC), ormass spectrometry; reaction mixtures were commonly purified by flashcolumn chromatography on silica gel, or by preparative HPLC using thegeneral protocol described below; NMR samples were dissolved indeuterated solvent (CD₃OD, CDCl₃, or DMSO-d6), and spectra were acquiredwith a Varian Gemini 2000 instrument (300 MHz) under standardparameters; and mass spectrometric identification was performed by anelectrospray ionization method (ESMS) with a Perkin Elmer instrument (PESCIEX API 150 EX).

Example 1 Synthesis of8-hydroxy-5-[(R)-1-hydroxy-2-(6-phenethylamino-hexylamino)ethyl]-1H-quinolin-2-onea. Preparation of (6-phenethylaminohexyl)-carbamic acid tert-butyl ester

(6-Aminohexyl)carbamic acid tert-butyl ester hydrochloride (5 g, 20mmol) was dissolved in 50% dichloromethane/methanol (50 mL) and treatedat ambient temperature with phenylacetaldehyde (2.3 mL, 20 mmol) for onehour. Sodium cyanoborohydride (1.2 g, 19 mmol) was added and the mixturestirred overnight. Water was added, followed by saturated sodiumhydrogencarbonate and additional dichloromethane. The mixture waspartitioned, the organics dried over sodium sulfate and the volatilesremoved under reduced pressure to give the title intermediate, which wasused without further purification.

b. Preparation of (6-tert-butoxycarbonylamino-hexyl)phenethylcarbamicacid benzyl ester

The product of the previous step was treated at ambient temperature withbenzylchloroformate (2.8 mL, 20 mmol) and aqueous sodium hydroxide (1 M,30 mL) overnight. The mixture was extracted with isopropyl acetate. Theorganics were washed with aqueous citric acid (0.5 M) followed bysaturated sodium hydrogencarbonate, dried over sodium sulfate and thevolatiles removed under reduced pressure to give the title intermediate(9 g) which was used without further purification.

c. Preparation of (6-aminohexyl)phenethylcarbamic acid benzyl ester

The product of the previous step (9 g crude) was dissolved indichloromethane (45 mL) and treated at ambient temperature withtrifluoroacetic acid (45 mL). The volatiles were removed under reducedpressure and the residue taken up in water and basified with sodiumhydroxide. The mixture was extracted with dichloromethane and theorganics dried over sodium sulfate. The volatiles were removed underreduced pressure to yield the title intermediate, which was evaporatedto dryness and used without further purification.

d. Preparation of[(R)-2-(tert-butyldimethylsilanyloxy)-2-(8-benzyloxy-2-oxo-1,2-dihydro-quinolin-5-yl)ethyl]-(6-phenethylamino-hexyl)carbamicacid benzyl ester

The product of the previous step (6.5 g crude, 18 mmol),8-benzyloxy-5-[(R)-2-bromo-1-(tert-butyldimethylsilanyloxy)ethyl]-1H-quinolin-2-one(4.5 g, 9.2 mmol) and dimethylsulfoxide (13 mL) were heated at 110° C.for one hour then cooled to ambient temperature overnight. The mixturewas partitioned between water and isopropyl acetate. The organics werewashed with saturated sodium hydrogencarbonate, dried over sodiumsulfate and evaporated to dryness. The mixture was purified byreverse-phase HPLC to give the title intermediate as its TFA salt.

e. Preparation of[(R)-2-hydroxy-2-(8-benzyloxy-2-oxo-1,2-dihydro-quinolin-5-yl)ethyl]-(6-phenethylamino-hexyl)carbamicacid benzyl ester

The product of the previous step was dissolved in tetrahydrofuran andtreated with triethylamine trihydrofluoride at room temperature untilthe reaction was adjudged to be complete by HPLC. The mixture waspartitioned between 1 M sodium hydroxide and isopropyl acetate. Theorganics were dried over sodium sulfate and evaporated to dryness togive the title intermediate.

f. Synthesis of8-hydroxy-5-[(R)-1-hydroxy-2-(6-phenethylamino-hexylamino)ethyl]-1H-quinolin-2-one

The product of the previous step (250 mg, 0.39 mmol) was dissolved indichloromethane at room temperature, and boron trichloride (1 M indichloromethane) was added in aliquots. The reaction was monitored byHPLC with additional boron trichloride being added until the reactionwas adjudged to be complete. 10% Acetic acid in water was added and thedichloromethane removed under reduced pressure. The residue was purifiedby reverse phase HPLC to give the title compound as itsbis-trifluoroacetate salt.

Example 2 Alternative Synthesis of8-hydroxy-5-[(R)-1-hydroxy-2-(6-phenethylamino-hexylamino)ethyl]-1H-quinolin-2-onea. Preparation of (5-phenethylcarbamoyl-pentyl)carbamic acid tert-butylester

Phenethylamine (340 μL, 4.3 mmol), 6-tert-butoxycarbonylamino-hexanoicacid (1.0 g, 4.3 mmol) and 1-hydroxybenzotriazole (700 mg, 5.1 mmol)were dissolved under nitrogen in N,N-dimethylformamide and cooled to 0°C. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was addedand the mixture stirred overnight, allowing to warm to room temperature.The mixture was partitioned between water and isopropyl acetate. Theorganics were washed with 0.5 M citric acid, then saturated sodiumhydrogen carbonate, dried over sodium sulfate and evaporated to dryness.The residue was used without further purification.

b. Preparation of 6-amino-N-(2-phenethyl)hexanamide

The product of the previous step was dissolved in dichloromethane (10mL) and treated with trifluoroacetic acid (10 mL) at room temperaturefor 1 hour. The volatiles were removed under reduced pressure and theresidue taken up in dichloromethane, washed with 1 N sodium hydroxide,dried over sodium sulfate and evaporated to dryness to give the titleintermediate (830 mg, 3.5 mmol) that was used without furtherpurification.

c. Preparation of N¹-phenethylhexane-1,6-diamine

The product of the previous step (330 mg, 1.4 mmol) was dissolved atroom temperature under nitrogen in tetrahydrofuran (4 μL) andborane-methyl sulfide complex (500 μL) was added. The mixture wasrefluxed overnight then cooled in an ice bath. Methanol was addedcarefully and the volatiles removed under reduced pressure. The residuewas redissolved in methanol and concentrated again to give the titleintermediate (360 mg, 1.6 mmol) which was used without furtherpurification.

d. Preparation of8-benzyloxy-5-[(R)-1-(tert-butyldimethylsilanyloxy)-2-(6-phenethylamino-hexylamino)ethyl]-1H-quinolin-2-one

The product of the previous step (200 mg, 0.91 mmol),8-benzyloxy-5-[(R)-2-bromo-1-(tert-butyldimethylsilanyloxy)ethyl]-1H-quinolin-2-one(340 mg, 0.70 mmol), sodium hydrogen carbonate (180 mg, 2.1 mmol) anddimethyl sulfoxide (1.7 mL) were heated to 110° C. for 55 minutes,cooled to room temperature and partitioned between isopropyl acetate andwater. The organics were dried over sodium sulfate and evaporated todryness. The residue was purified by reverse phase HPLC to give thetitle intermediate as its bis-trifluoroacetate salt (75 mg, 88 μmol).

e. Preparation of8-benzyloxy-5-[(R)-1-hydroxy-2-(6-phenethylamino-hexylamino)ethyl]-1H-quinolin-2-one

The product of the previous step (75 mg, 88 μmol) was dissolved intetrahydrofuran (1 mL) and treated with triethylamine trihydrofluoride(60 μL, 370 μmol) at room temperature for 18 hours. The mixture ispartitioned between 1M sodium hydroxide and isopropyl acetate. Theorganics are dried over sodium sulfate and evaporated to dryness to givethe title intermediate.

f. Synthesis of8-hydroxy-5-[(R)-1-hydroxy-2-(6-phenethylamino-hexylamino)ethyl]-1H-quinolin-2-one

The product of the previous step was dissolved in dichloromethane atroom temperature and boron trichloride (1 M in dichloromethane) wasadded in aliquots. The reaction was monitored by HPLC with additionalboron trichloride being added until the reaction was adjudged to becomplete. 10% Acetic acid in water was added and the dichloromethaneremoved under reduced pressure. The residue was purified by reversephase HPLC to give the title compound as its bis-trifluoroacetate salt.m/z: [M+H⁺] calcd for C₂₅H₃₃N₃O₃: 424.26; found 424.4. ¹H NMR (300 MHz,DMSO-d₆): 10.4 (s, 2H), 8.6 (br s, 4H), 8.0 (d, 1H, J=9.9 Hz), 7.1-7.3(m, 5H), 7.0 (d, 1H, J=8.2 Hz), 6.9 (d, 1H, J=8.2 hz), 6.4 (d, 1H, J=9.9Hz), 6.1 (br s, 1H), 5.2 (br d, 1H, J=8.5 Hz), 2.7-3.1 (m, 10H), 1.4-1.6(m, 4H), 1.1-1.3 (m, 4H).

Comparison Example Synthesis of8-hydroxy-5-[(R)-1-hydroxy-2-(6-phenethyloxy-hexylamino)ethyl]-1H-quinolin-2-onea. Preparation of 6-phenethyloxyhexanenitrile

6-Bromohexanenitrile (3.0 mL, 23 mmol) and tetrabutylammonium bromide(350 mg, 1.1 mmol) were dissolved in tetrahydrofuran (5 mL) and 50%aqueous sodium hydroxide (15 mL). Phenethyl alcohol (3.0 mL, 25 mmol)was added resulting in a precipitate. The mixture was heated to 65° C.whereupon the solid dissolved. The mixture was stirred at 65° C. forfour hours then cooled to room temperature and left for 11 days. Water,hexanes and ethyl acetate were added and the mixture partitioned. Theorganics were washed with water, saturated sodium chloride, dried oversodium sulfate and evaporated to dryness to give the title intermediate(5.3 g, 24 mmol) which was used without further purification.

b. Preparation of 6-phenethyloxyhexylamine

The product of the previous step (5.3 g, 24 mmol) was dissolved intetrahydrofuran (50 mL) and treated with borane-methyl sulfide complex(6 mL). The mixture was refluxed for 3.5 hours, then additionalborane-methyl sulfide complex (2 mL) was added, the mixture was refluxedfor an additional 20 hours, cooled to room temperature, and methanol wasadded carefully. The volatiles were removed under reduced pressure andthe mixture re-evaporated from methanol to give the title intermediate(5.7 g, 26 mmol), which was used without further purification.

c. Preparation of8-benzyloxy-5-[(R)-1-(tert-butyldimethylsilanyloxy)-2-(6-phenethyloxy-hexylamino)ethyl]-1H-quinolin-2-one

The product of the previous step (3.4 g, 15 mmol),8-benzyloxy-5-[(R)-2-bromo-1-(tert-butyldimethylsilanyloxy)ethyl]-1H-quinolin-2-one(3.4 g, 7.0 mmol), sodium hydrogen carbonate (2.3 g, 27 mmol) anddimethylsulfoxide (7 mL) were heated at 100° C. for 135 minutes thencooled to room temperature and allowed to stand for 16 hours. Themixture was partitioned between water and isopropyl acetate and theorganics washed with water then with saturated sodium chloride, driedover sodium sulfate and evaporated to dryness. The product was purifiedby reverse phase HPLC to give the title intermediate as itstrifluoroacetate salt (1.5 g, 2.0 mmol).

d. Preparation of8-benzyloxy-5-[(R)-1-hydroxy-2-(6-phenethyloxy-hexylamino)ethyl]-1H-quinolin-2-one

The product of the previous step (1.5 g, 2.0 mmol) was dissolved intetrahydrofuran (15 mL) and treated with triethylamine trihydrofluoride(660 μL, 4.0 mmol) at room temperature for 3 days. The mixture waspartitioned between 1 M sodium hydroxide and ethyl acetate. The organicswere washed with saturated sodium chloride, dried over sodium sulfateand evaporated to dryness to give the title intermediate (1.4 g, 2.7mmol), which was used without further purification.

e. Synthesis of8-hydroxy-5-[(R)-1-hydroxy-2-(6-phenethyloxy-hexylamino)ethyl]-1H-quinolin-2-one

The product of the previous step (1.4 g, 2.7 mmol) and palladiumhydroxide on carbon (20% Pd, moisture content ˜60%, 300 mg) weresuspended in tetrahydrofuran (30 mL) and stirred under a hydrogenatmosphere at room temperature for two hours. The palladium was removedby filtration and the mixture evaporated under reduced pressure. Theresidue was purified by reverse-phase HPLC to give the title compound asits trifluoroacetate salt. m/z: [M+H⁺] calcd for C₂₅H₃₂N₂O₄: 425.25;found 425.3. ¹H NMR (300 MHz, DMSO-d₆): 10.4 (s, 1H), 10.4 (s, 1H), 8.4(br s, 2H), 8.0 (d, 1H, J=10.2 Hz), 7.0-7.2 (m, 6H), 6.9 (d, 1H, J=8.0Hz), 6.5 (d, 1H, J=9.9 Hz), 6.1 (d, 1H, J=3.7 Hz), 5.2 (m, H), 3.4 (t,2H, J=7.0 Hz), 3.2 (t, 2H, J=6.6 Hz), 2.7-3.0 (m, 4H), 2.7 (t, 2H, J=6.9Hz), 1.3-1.5 (m, 4H), 1.1-1.2 (m, 4H).

Example 4 Biological Assay Results

A representative compound of the invention, the compound of Examples 1and2,8-hydroxy-5-[(R)-1-hydroxy-2-(6-phenethylamino-hexylamino)ethyl]-1H-quinolin-2-oneand the compound of the Comparison Example,8-hydroxy-5-[(R)-1-hydroxy-2-(6-phenethyloxy-hexylamino)ethyl]-1H-quinolin-2-one,were tested in the biological assays described above. Results of theassays are summarized in the following tables. TABLE 1 In vitro AssayResults Compound of Comparison the Invention Compound Binding AffinityAssay (Test A) K_(i)(β₂) 7.6 9.1 Selectivity β₂/β₁ 18 76 FunctionalAssay (Test B) Recombinant Cell Line pEC₅₀ 9.5 10.3 Selectivity β₂/β₁107 54 Selectivity β₂/β₃ 3900 370 Functional Assay (Test C) EndogenousCell Line pEC₅₀ 8.5 % Efficacy 68

TABLE 2 In vivo Assay Results Guinea Pig Bronchoprotection Compound ofComparison Model (Test D) the Invention* Compound Activity at 1.5 hourspost dose Yes Yes Activity at 72 hours post dose Yes No*Compound prepared in Example 1

The tested compound of the invention was active at the β₂ adrenergicreceptor in the in vitro binding affinity and functional assaysdescribed above, as was the comparison compound. Surprisingly, thecompound of the invention demonstrated bronchoprotection in the guineapig model at 72 hours post dose, while the comparison compound was notactive in vivo at 72 hours post dose. Furthermore, the compound of theinvention showed excellent selectivity for the β₂ adrenergic receptor ascompared with the β₁ and β₃ receptor subtypes in the functional assay ofTest B. The assay results indicate the invention provides potent andselective β₂ adrenergic receptor agonists having exceptionally longduration of action.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto. Additionally, all publications, patents, andpatent documents cited hereinabove are incorporated by reference hereinin full, as though individually incorporated by reference.

1-29. (canceled)
 30. A compound of formula (II):

wherein: R¹ is —CH₂OH or —NHCHO, and R² is hydrogen; or R¹ and R² taken together are —NHC(═O)CH═CH— or —CH═CHC(═O)NH—; n is an integer of from 2 to 6; m is an integer of from 0 to 3; or a pharmaceutically-acceptable salt or solvate or stereoisomer thereof.
 31. The compound of claim 30 wherein n is
 4. 32. The compound of claim 30 wherein m is
 1. 33. The compound of claim 30 wherein R¹ and R² taken together are —NHC(═O)CH═CH— or —CH═CHC(═O)NH—.
 34. The compound of claim 30 wherein the stereochemistry at the alkylene carbon bearing the hydroxyl group is (R).
 35. The compound of claim 30 having the chemical name 8-hydroxy-5-[(R)-1-hydroxy-2-(6-phenethylamino-hexylamino)ethyl]-1H-quinolin-2-one; or a pharmaceutically-acceptable salt or solvate or stereoisomer thereof.
 36. A pharmaceutical composition comprising a therapeutically effective amount of the compound of claim 30 and a pharmaceutically acceptable carrier.
 37. The pharmaceutical composition of claim 36, wherein the composition further comprises a therapeutically effective amount of one or more other therapeutic agents selected from a corticosteroid, an anticholinergic agent, and a PDE4 inhibitor.
 38. The pharmaceutical composition of claim 36, wherein the composition is formulated for administration by inhalation.
 39. A pharmaceutical composition comprising a therapeutically effective amount of the compound of claim 35 and a pharmaceutically acceptable carrier.
 40. The pharmaceutical composition of claim 39, wherein the composition is formulated for coadministration with 6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioic acid S-fluoromethyl ester.
 41. A combination comprising the compound of claim 30 and one or more other therapeutic agents selected from a corticosteroid, an anticholinergic agent, and a PDE4 inhibitor.
 42. A combination comprising the compound of claim 35 and 6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioic acid S-fluoromethyl ester.
 43. A process for preparing a compound of formula (II):

wherein: R¹ is —CH₂OH or —NHCHO, and R² is hydrogen; or R¹ and R² taken together are —NHC(═O)CH═CH— or —CH═CHC(═O)NH—; n is an integer of from 2 to 6; m is an integer of from 0 to 3; or a pharmaceutically-acceptable salt or stereoisomer thereof, the process comprising: (a) reacting a compound of formula 1

wherein: P¹ and P² are each a hydroxy-protecting group and L is a leaving group, with a compound of formula 2:

wherein R¹¹ is hydrogen or P³ wherein P³ is an amino-protecting group, to provide a compound of formula 3:

(b) removing the protecting group P¹ to provide a compound of formula 4:

(c) removing the protecting group P² and the protecting group P³, if present, to provide a compound of formula (II), or a salt or stereoisomer thereof.
 44. The product prepared by the process of claim
 43. 45. A method of treating a mammal having a disease or condition associated with β2 adrenergic receptor activity, the method comprising administering to the mammal, a therapeutically effective amount of the compound of claim
 30. 46. The method of claim 45 further comprising administering a therapeutically effective amount of one or more other therapeutic agents selected from a corticosteroid, an anticholinergic agent, and a PDE4 inhibitor.
 47. A method of treating a mammal having a disease or condition associated with β₂ adrenergic receptor activity, the method comprising administering to the mammal, a therapeutically effective amount of the compound of claim 35 and 6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioic acid S-fluoromethyl ester.
 48. The method of claim 47 wherein the method comprises administering the compound by inhalation.
 49. A method of treating asthma or chronic obstructive pulmonary disease in a mammal, the method comprising administering to the mammal, a pharmaceutical composition comprising a pharmaceutically-acceptable carrier and a therapeutically effective amount of the compound of claim
 35. 